Objective To examine transcription levels of vitamin D metabolic enzymes and the vitamin D receptor (VDR) in the testes of adult tree shrews. Specifically, to detect protein expression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) and investigate causes of reproductive disorders in male tree shrews. Methods Male tree shrews (aged 2 to 3 years) were selected. On the basis of individual records, shrews were divided into a normal group with a successful reproductive history and a reproductive disorder group without a reproductive history; there were five shrews in each group. One side of the testis was surgically removed and histopathological changes were observed by pathology. Real- time fluorescent quantitative PCR was used to detect changes in mRNA levels of VDR and vitamin D metabolizing enzymes in the testis. Protein expression of CYP24A1 was detected by western blotting. Expression of sperm CYP24A1 was detected by immunofluorescence. Results In the normal group, the seminiferous tubules were tightly arranged and there were more spermatogenic cells than in the reproductive disorder group. Compared with the normal group, there was no significant difference in mRNA levels of VDR, CYP2R1, or CYP27A1 in the testes of tree shrews (P> 0. 05). However, mRNA expression levels and protein levels of CYP24A1 were significantly lower than in the normal group (P< 0. 05). At the same time, immunofluorescence showed that sperm from the testis of the normal group had high expression of CYP24A1 in the head, neck, and midpiece, while the reproductive disorder group had low or no expression of CYP24A1. Conclusions CYP24A1 is directly related to successful fecundity of male tree shrews, suggesting that CYP24A1 can be used as an important index to evaluate the fecundity of male tree shrews.